PF was from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany), with a purity >98%. The molecular formula of PF is C₂₃H₂₈O₁₁, and its molecular weight is 480.46. PF was dissolved in PBS to produce a stock solution. The PF stock solution was diluted in a cell culture medium prior to its use in each experiment. Trypsin (0.25%), PBS, fetal bovine serum (FBS), Eagle's Minimum Essential medium (EMEM) and Mcroy' 5A medium were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Hoechst 33258 and an MTS kit were from Promega Corporation (Madison, WI, USA). Antibodies against caspase-3 (cat. no. 9662), cleaved-caspase-3 (cat. no. 9661), poly (ADPribose) polymerase (PARP; cat. no. 9532), cleaved-PARP (cat. no. 5625), BH3 interacting domain death agonist (Bid; cat. no. 2002), Bcl-2 (cat. no. 4223), Bcl-2 X-associated protein (Bax; cat. no. 5023), Bcl-extra large (XL; cat. no. 2764), cyclin B1 (cat. no. 4135), p21 (cat. no. 2947) and β-actin (cat. no. 3700) were from Cell Signaling Technology, Inc., (Beverly, MA, USA). Antibodies against cyclin-dependent kinase (CDK)1 (cat. no. ab133327) and phosphorylated (p)-CDK1 (p-Y15; cat. no. ab133463) were purchased from Abcam (Cambridge, UK).

The HOS [CRL-1547TM, American Type Culture Collection, Manassas, VA, USA (ATCC)] and Saos-2 (HTB-85TM; ATCC) human osteosarcoma cell lines were from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). HOS cells were cultured in EMEM and Saos-2 cells were cultured in Mcroy' 5A medium containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). All cell lines were cultured at 37°C in a 5% (v/v) CO₂ humidified incubator. The present study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University Medical School (Zhejiang, China).

The viability of osteosarcoma cells treated with PF was measured by an MTS assay. In brief, cells were seeded into 96-well plates (5,000–6,000 cells/well) overnight, then they were treated with PF at concentrations ranging from 200 to 500 µM for 0–48 h. The MTS kit was added and cells were incubated at 37°C in a humidified incubator for 1–3 h following the manufacturer's protocol. Absorbance was read on a MR7000 microplate reader (Dynatech Nevada, Inc., Carson City, NV, USA) at 490 nm and the probit model was used to obtain IC50 values. Data were averaged in six replicates. Each assay was tested in triplicate.

To assess the characteristic morphological changes of osteosarcoma cell apoptosis, Hoechst 33258 staining followed by fluorescent microscopy was performed. Cells were treated with PF for 24 h. Subsequently, cells were stained with Hoechst 33258 at room temperature for 10 min. Subsequently, cells were washed twice with PBS and observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) to identify chromatin condensation and nuclei fragmentation.

The morphological ultra-structure changes of treated and control cells were observed using transmission electron microscopy (TEM). Cells were fixed with 2.5% glutaraldehyde at 4°C for 4 h and post-fixed in 1% osmium tetroxide at 4°C for 1 h. The cell pellets were embedded in epoxy resin following dehydration in different concentrations of alcohol. Sections (0.5-µm) were treated with uranyl acetate and lead citrate, then observed at a magnification of ×8,300 under a transmission electron microscope.

Apoptosis caused by PF was detected by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining (Biouniquer Technology, Nanjing, China). In brief, cells were incubated in six-well plates at a density of 2×10⁵ cells/well and treated with varying concentrations of PF (0–500 µM) for 24 h. The cells were stained using Annexin V-FITC (5 µl) and PI (5 µl) for 15 min in the dark. A flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) and ModFit LT (version 3.3; Verity Software House, Topshame, ME, USA) was used to analyze the samples.

PI/RNase staining buffer (BD Biosciences) was used to determine cell cycle with flow cytometry. In brief, after incubation with different concentrations of PF (0, 200, 300 and 500 µM) for 24 h, ~5×10⁵ cells were fixed with 70% ethanol at −20°C for 24 h. Following this, the cells were incubated with PI/RNase and tested with a flow cytometer (FACSCalibur).

Cells (5×10⁵) were incubated in 60-mm dishes and treated with PF for 24 h. Protein lysates were obtained by lysing cells with radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA) containing a mixture of protease inhibitors (Sigma-Aldrich; Merck KGaA) for 0.5 h at 4°C to extract total proteins. Next, a bicinchoninic acid total protein quantitation kit was used to identify the protein content. Total proteins (60 µg) were separated by electrophoresis on a 5% stacking gel and an 8–12% separating gel. Proteins were transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA) and blocked with 5% bovine serum albumin at 20°C for 1 h. Membranes were incubated with primary antibodies (1:1,000) at 4°C for 12 h. After that, the membranes were washed five times with TBS with 0.1% Tween 20 and then incubated with goat anti-rabbit and anti-mouse immunoglobulin G horseradish peroxide conjugated secondary antibodies (Pierce; Thermo Fisher Scientific, Inc.; 1:5,000) at 37°C for 1 h. Following this, an Enhanced Chemiluminescence kit was used to visualize proteins with exposure to X-ray film (Kodak, Rochester, NY, USA). BandScan 5.0 software (Glyko, Inc., Novato, CA, USA) were used to analyze the density of strips. The relative expression amount of the target protein is expressed as: (Objective protein optical density value)/(β-actin optical density value) ×10.

All data are expressed as the mean ± standard deviation. The experiments were triplicated to obtain the mean values. The statistical differences were calculated by unpaired Student's t-test or one-way analysis of variance followed by Dunnett's test using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To study the effects of PF on the growth of osteosarcoma cells, an MTS assay was used to measure the cell viability of HOS and Saos-2 cells. As the concentration of PF increased, cell viability decreased; following incubation with PF for 48 h, the IC50 values of PF were 363.29 µM for HOS and 351.24 µM for Saos-2 (Fig. 1A). The results indicated that PF has the capability to inhibit cell viability in a dose- and time-dependent manner.

In order to demonstrate whether PF inhibits cell proliferation through mediating cell cycle arrest, flow cytometry analyses were used to examine the distribution of the cell cycle. As presented in Fig. 1B and C, after incubating with PF for 48 h, HOS and Saos-2 cells exhibited a significant increase in G2/M phase arrest, with a corresponding decline in the G0/G1 and S phases in a dose-dependent manner. To determine the underlying mechanism, the protein expression levels of cyclin B1, CDK1, p-CDK1 and p21, which have been demonstrated to be cell cycle-regulating proteins, were assessed. The protein expression levels of cyclin B1, p-CDK1, CDK1 and p21 increased after treating with PF in HOS and Saos-2 cells in a dose-dependent manner (Fig. 1D and E).

To investigate the inhibition mechanism of PF in the proliferation of osteosarcoma cells, Hoechst 33258, TEM and flow cytometry analyses were used in the present study. The Hoechst 33258 staining revealed morphological changes when the cells were treated with PF, such as karyopyknosis and nuclear fragmentation (Fig. 2A). Furthermore, TEM revealed more detailed apoptotic morphological features in cells treated with PF compared with control cells, including cell shrinkage, a denser cytoplasm, cytoplasmic vacuoles, karyopyknosis and the presence of apoptotic bodies (Fig. 2B). Next, flow cytometry analysis was used to quantify PF-induced apoptosis in human osteosarcoma cells. PF led to apoptosis in osteosarcoma cell lines in a dose-dependent manner, consistent with the results of the MTS assay. Following treatment with PF, Saos-2 cells exhibited increased rates of apoptosis from 6.3% at 0 µM to 48.7% at 500 µM, and HOS cells from 8.8% at 0 µM to 43.8% at 500 µM (Fig. 2C and D). The result was consistent with the finding demonstrated by the MTS assay.

To detect the potential cell signaling pathways in apoptosis induced by PF, the protein expression levels of the caspase-3, PARP and Bcl-2 family were examined (Fig. 3). The expression levels of Bcl-2 and Bcl-XL protein were downregulated following PF treatment in a dose-dependent manner, in both cell lines. However, the levels of Bax and Bid protein were upregulated after incubation with PF for 48 h (P<0.05). Additionally, the levels of cleaved caspase-3 and cleaved PARP protein were significantly upregulated in a dose-dependent manner (P<0.05). Therefore, these results demonstrated that apoptosis induced by PF occurred via downregulation of the anti-apoptotic proteins Bcl-XL and Bcl-2 and upregulation of the pro-apoptotic proteins Bax, Bid, PARP and caspase-3 (Fig. 4).